In vitro selection of desired biological macromolecules from a pool of available biological macromolecules has become a useful tool in for instance the research on molecular interactions, medical imaging including diagnosis, or in the generation of protein-based biopharmaceuticals including recombinant antibodies. In vitro display technology for the selection of peptides and proteins relies on a physical linkage between the peptide or protein and a nucleic acid encoding the same. A large panel of techniques has been established for this purpose, with the most commonly used being phage/virus display, ribosome display, cell-surface display, ‘peptides on plasmids’, mRNA display, DNA display, cDNA display and in vitro compartmentalisation including micro-bead display (for reviews see e.g. Rothe, A., et al., FASEB J. (2006) 20, 1599-1610; Sergeeva, A., et al., Advanced Drug Delivery Reviews (2006) 58, 1622-1654).
Display techniques allow the generation of engineered antibodies and ligands with high affinities for a selected target molecule. It is thus also possible to display an array of peptides or proteins that differ only slightly, typically by way of genetic engineering. Thereby it is possible to screen and subsequently evolve proteins or peptides in terms of properties of interaction and biophysical parameters. Iterative rounds of mutation and selection can be applied on an in vitro basis.
Different means of physically linking the protein or peptide and the respective nucleic acid have been disclosed. Expression in a cell with a cell surface molecule, expression as a fusion polypeptide with a viral/phage coat protein, a stabilised in vitro complex of an RNA molecule, the ribosome and the respective polypeptide, covalent coupling in vitro via a puromycin molecule or via micro-beads are examples of ways of linking the protein/peptide and the nucleic acid presently used in the art.
A further technique of linking a protein or peptide and the respective nucleic acid that does not involve the formation of a physical linkage relies on a water-in-oil emulsion. The water droplets serve as compartments in each of which a single gene is transcribed and translated (Tawfik, D. S., & Griffiths, A. D., Nature Biotech. (1998) 16, 652-656, US patent application 2007/0105117). This physical linkage between the peptide or protein and the nucleic acid (encoding it) provides the possibility of recovering the nucleic acid encoding the selected protein or peptide. A nucleic acid with a respective gene and a corresponding protein are clonally constrained within a compartment. Compared to techniques such as immunoprecipitation, in display techniques thus not only binding partners of a selected target molecule can be identified or selected, but the nucleic acid of this binding partner can be recovered and used for further processing.
A further technique for the selection of desired proteins is the use of the yeast two-hybrid system with vectors having a Lox site and the recombinase Cre (Hastie, A. R., & Pruitt, S. C., Nucleic Acids Research (2007) 35, 12, e141).
Present display techniques thus provide means for e.g. target discovery, lead discovery and lead optimisation. Vast libraries of peptides or proteins, e.g. antibodies, potentially can be screened on a large scale. However, in order to be able to isolate a complex between a protein or peptide and a target molecule and to remove low-affinity binders, immobilisation of one of the two binding partners is required. Thus current display techniques depend on immobilising the target molecule prior to analysis, typically on the surface a multi-well plate used in the screening process or, in case of in vitro compartmentalisation micro-beads.
Furthermore, current display techniques are one-dimensional in that they require providing a single target molecule against which a broad spectrum of (potentially binding) peptides or proteins can be tested. Simultaneous testing of different target molecules would require isolating and analysing both the respective target and its binding partner and is therefore not practicable.
Accordingly it is an object of the present invention to provide a method of identifying binding partners that can be applied to both immobilised analytes and analytes in solution. As indicated above, such a method would also drastically improve the flexibility of in vitro display technology.